Bilirubin is a breakdown product of hemoglobin which is an oxygen carrier in blood. In human body bilirubin is present in two forms. Indirect or unconjugated bilirubin (insoluble in water) is present as free bilirubin that circulates in the plasma and is taken up by liver cells where it is enzymatically esterified with glucuronic acid to form conjugated or direct bilirubin. Conjugated bilirubin is water soluble and is easily excreted through bile. The normal levels of bilirubin in human blood is as follows; direct bilirubin in the range of 0 to 0.3 mg/dl and total bilirubin should be 0.3 to 1.9 mg/dl. Abnormal levels of total bilirubin (conjugated and unconjugated) detected in serum samples is an indicator for disturbed bilirubin metabolism. Determination of the amount of bilirubin in body fluid, especially in blood, is important factor for detection of haemolysis and for checking liver function. The excess or over production or problem in bilirubin metabolism leads to jaundice, liver disorders etc. Therefore, detecting bilirubin levels in the body is very important to regulate our body. Diazotization is one of the most common methods of determination, wherein, bilirubin is coupled with diazonium salt such as diazosulfanilic acid and the amount of the resulting colorant is measured in a spectrophotometer to estimate the bilirubin content in the sample. Details of the diazotization method are described in J. B. Landis and R L. Prude, Clinical Chemistry, 24 (10), 1690-1699 (1978). However, this method has its own disadvantage as it take lots of time for total color development and also overestimates the amount of conjugated bilirubin.
U.S. Pat. No. 4,612,290 discloses a method for quantitative determination of bilirubin which comprises bringing a bilirubin-containing aqueous liquid sample into contact with a hydrophobic bilirubin extracting composition containing a hydrophobic amine capable of extracting bilirubin. The amine extracts the bilirubin in said aqueous liquid sample. Photometry is then used to determine the concentration of bilirubin extracted with the bilirubin extracting composition.
Article titled “Self-Assembly in Tailor-Made Polyfluorenes: Synergistic Effect of Porous Spherical Morphology and FRET for Visual Sensing of Bilirubin” by T. Senthilkumar and S. K. Asha published in Macromolecules, 2013, 46 (6), pp 2159-2171 reports two new fluorene-based homo-(PDP-PF) and copolymers (PDPPF-co-Ph) were synthesized with a bulky 3-pentadecylphenoxy (PDP) group appended hexyl chains at the 9, 9′ position using Suzuki coupling polymerization. Also the sensing efficiency of both polymers toward the biologically important analyte bilirubin was demonstrated in organic medium by the quenching of polymer fluorescence and FRET-based bilirubin emission.
Article titled “Detection and measurement of total bilirubin in serum, with use of surfactants as solubilizing agents” published in Clinical Chemistry, 1974; 20(4), 447-53 reports a new method for measuring total bilirubin in serum. Nonionic, cationic, or anionic surfactants can be used as solubilizing agents to promote the diazo coupling of indirect-reacting bilirubin.
Dissertation titled” Ionic, Water-Soluble Polyfluorene-Type Copolymers” by swapna Pradhan reports the synthesis of 2,7-dibromo-9,9-bis(6-bromohexyl)fluorene and Synthesis of poly {9,9-bis[6-(N,N-dimethylamino)hexyl]fluoreneco-1,4-phenylene}. The report also illustrated the conjugated copolymers based on alternating fluorene and phenylene building blocks are also promising efficient and stable blue luminescent materials.
In spite of the above and other existing methods, sensing bilirubin is a very challenging task because of its less solubility, poor emission property and poor quantum yield in water as well as in common organic solvents. Conjugated polymers are used in chemo and biosensors and they can be structurally modified to improve water solubility, good emission and high quantum yield. For the present invention polyfluorene was functionalized with glucuronic acid which made it water soluble. Glucuronic acid is known for its ability for selective interaction with bilirubin. Thus, functionalizing polyfluorene with glucuronic acid has the added advantage of introducing secondary interactions between bilirubin and glucuronic acid, which is expected to bring the polymer and bilirubin closer for better and more efficient fluorescence sensing response.
The delocalised π electrons in conjugated polymers are sensitive even to minor perturbations resulting in amplified signal response due to which they find applications as chemical and biosensors. By taking advantage of overlapping photophysical properties of bilirubin and polyfluorenes the inventors have developed a model polyfluorene sensor to detect unconjugated bilirubin via FRET process for the first time. A few attempts have been reported in literature for the sensing of bilirubin in aqueous medium via the fluorescence technique. However, despite the best efforts, sensing of bilirubin in water is very difficult, due to poor solubility, reactivity and very low quantum yield of bilirubin emission. The other challenge is the selectivity in the sensing processes.
Therefore, designing and synthesizing a fluorescence sensor with high selectivity, and detecting the bilirubin in aqueous medium is desired.